expressed in nodules, but not in uninfected roots. Intensive studies over the past two decades have
identified almost 200 nodulin genes in different plant hosts [6]. Nodulins are defined as early or
late according to the time of appearance during nodule development. Early nodulins are expressed
well before nitrogen fixation onset and function in nodule development and infection processes [3,5].
Although the expression of several early nodulin genes has been well documented, the exact roles of
the encoded proteins are still unknown [7 – 11]. Late nodulins participate in nodule function and
appear in nodules during the onset of nitrogen fixation, after the nodule structure has developed
[3,5]. This group of nodulins includes leghemo- globins; enzymes functioning in nitrogen and car-
bon metabolism glutamine synthetase, uricase, sacharose synthatese; peribacteroid membrane
proteins and many others, of which the function remains to be elucidated [3,12 – 16]. Recently, it
has
been reported
that the
Vicia sati6a
leghemoglobin gene Vslb
1
, is expressed in root hairs within 1 h after Nod factor treatment indi-
cating that the function of some nodulins may have to be revised [17].
Lupine root nodules have been described as a unique lupinoid subtype within the indeterminate
class [18]. The meristem is located basi-laterally and contains bacteroids, so there is no distinctly
marked infection zone. Initial cell divisions occur hypodermally. During nodule growth the connec-
tion to the root broadens causing the nodule to surround the root [18].
Several lupine nodulin genes have been iden- tified and characterized. These include genes cod-
ing for two leghemoglobins LbI and LbII, glutamine synthetase and ENOD2-like protein
[19,20]. To identify additional nodulin genes and to increase the repertoire of usable molecular
probes, the differential display approach was em- ployed [21,22]. Differential display has been suc-
cessfully used to identify new nodulin genes from Sesbania rostrata and to prepare a nodulin ex-
pressed sequence tag library from Lotus japonicus [23,24]. In this study expression analysis of several
newly identified nodulin genes during lupine nod- ule development is presented. One of the isolated
clones encodes a putative stearoyl-acyl carrier protein desaturase. A function for this enzyme in
nodule development is proposed.
2. Material and methods
2
.
1
. Plant material Yellow lupine Lupinus luteus cv. Ventus seeds
were surface sterilized in 0.1 HgCl
2
and 0.01 Tween 20 for 15 min, washed intensively with
sterile water and germinated for 3 days at 28°C in Petri dishes in full darkness. Seedlings were
moved to perlite and watered with 7 ml of a freshly grown culture of Bradyrhizobium sp.
Lupinus USDA 3045 OD = 0.5 – 0.6. Plants were kept in a growth room with a 16 h
photoperiod at 25°C and 8 h dark at 20 – 22°C. Nodules were harvested 6, 9, 13, 15, and 21 days
after inoculation DAI. Uninfected roots, leaves and stems were cut 21 DAI and fruits were
harvested from plants 60 days after sowing. During growth, plants were watered with a
medium containing micro- and macroelements, but the medium for nodulating plants lacked
inorganic nitrogen [19].
2
.
2
. RNA isolation and northern hybridization Total RNA was isolated essentially according to
Ref. [25], although three extractions with phenol were
required to
obtain RNA
with A260
A280 \ 1.8. RNA was dissolved in DEPC-treated water and kept at − 70°C. Integrity of RNA
was checked by formaldehyde agarose gel elec- trophoresis [26]. For northern hybridizations, 20
m
g samples of total RNA were separated elec- trophoretically
on denaturing
agarose gels,
stained in ethidium bromide solution and blotted to nitrocellulose filters according to the manufac-
turer’s protocol [Schleicher and Schuel]. DNA for probes was purified from agarose gels and
then labeled in a random priming reaction with 30 – 50
m Ci
of [a
32
P]dATP following
the manufacturer
’
s protocol
[Promega]. Labeled
probes were purified from unincorporated nucle- otides
on sephadex
G-50 fine
[Pharmacia] columns. Filters were hybridized for 24 – 72 h in
a standard solution containing 6 × SSC, 5 × Denhardt solution, 1 SDS, 50 formamide
and 70 mgml of denatured salmon sperm DNA. Filters were washed using high stringency condi-
tions 0.1 × SSC, 0.1 SDS, 60°C. If differential displayed probes were used, washing conditions
were less stringent 0.3 × SSC, 0.1 SDS, 50°C.
2
.
3
. Differential display RNA fingerprinting RNA differential display reactions were carried
out using a RNAmap kit GenHunter, Brooklyn MA. Half microgram samples of total RNA iso-
lated from nodules 21 days after infection with Bradyrhizobium sp. Lupinus USDA 3045 and
uninfected roots were reverse transcribed and 1 ml aliquots amplified according to the protocol pro-
vided by the manufacturer, although reactions were scaled down to 10 ml. PCR products were
separated on 6 polyacrylamide sequencing gels. After 48 h of autoradiography, nodule-specific
products were cut out from dried gels and the DNA eluted by boiling with 100 ml water and
precipitated with 3 volumes of ethanol in the presence
of 20
m g
glycogen [Boehringer
Mannheim]. Reamplification of 3 ends of cDNA fragments was performed according to standard
protocol 40 ml volume, 20 mM dNTPs, 1.5 mM MgCl
2
[21,22]. Reamplified fragments were blunt ended by a 15 min incubation at 37°C with 1U of
DNA Polymerase I Klenow fragment in the pres- ence of 50 mM dNTPs. DNA fragments were
separated on 2 agarose gels and, after purifica- tion, phosphorylated with T4 DNA kinase [Amer-
sham] and cloned into the SmaI site of pBluescipt SK-.
2
.
4
. cDNA library screening A yellow lupine nodule cDNA library con-
structed in a l-ZAP II vector Stratagene was screened with the [a
32
P]dATP-labeled differential display probes, prepared from individual cloning
products that showed nodule specificity in north- ern hybridization. Filters were hybridized at 55°C
for 36 – 48 h and then washed two times in 2 × SSC at room temperature, once at 55°C in 1 ×
SSC and two times in 0.2 × SSC, 0.1 SDS at 50°C.
2
.
5
. DNA sequencing and computer analysis cDNA clones were sequenced using a Sequenase
II kit USB Amersham, according to the manu- facture’s protocol. Homology searches were done
using the BLAST and FASTA algorithms. Se- quence
alignment was
performed using
the CLUSTALW
program. The
PROSITE and
BLOCKS databases were searched to identify con- served amino acid motifs. Prediction of the cleav-
age site in signal peptides was based on rules described by von Heijne [27].
2
.
6
. RT-PCR analysis Reverse transcription reactions were performed
according to
the manufacturer’s
instruction [Boehringer Mannheim]. Briefly, cDNAs were syn-
thesized from 3 mg total RNA isolated from nod- ules 6, 9, 13, 21 DAI, leaves, stems and young
fruits for 2 h at 37°C in reaction buffer containing 50 mM Tris – HCl pH 8.3, 40 mM KCl, 6 mM
MgCl
2
, 10 mM dithiotreitol, 200 ng of oligo dT18, 1 mM dNTPs. Reactions were stopped by heating
for 5 min at 95°C, diluted 50 times and 2 ml were used in PCR to amplify cDNA fragments using 35
cycles of amplification. Primers [MWG-Biotech] used in PCR and temperature conditions of am-
plification reactions were as follows: 5-atgggtgctt- taactgagagtcaagcag-3,
3-ctacttactacgacgtatta-5 94°C 30 s, 50°C 30 s, 72°C 40 s for amplification
of the yellow lupine leghemoglobin II fragment; 5-aagttgagccacataggt-3, 5-acaacactgataataaggca-
3 94°C 30 s, 45°C 30 s, 72°C 35 s for DD2T15 cDNA; 5-ggatttcacggatcaggtg-3, 5-cgagctgtgttgc-
catgtg-3 94°C 30 s, 50°C 30 s, 72°C 40 s for DD3A20 cDNA.
2
.
7
. Nucleotide sequence accession numbers The cDNA nucleotide sequences reported in this
paper have been deposited in GenBank under accession
numbers AF139377
DD3A20, AF139378
DD2A18, AF139379
DD4A9, AF139380 DD2T15-S and AF139381 DD2T15-
L.
3. Results
